A flavoring viscosity-reducing agent and its application in reconstituted tobacco concentrate

Abstract

The application discloses a flavoring and viscosity-reducing microbial agent and application thereof in reconstituted tobacco concentrate. The flavoring and viscosity-reducing microbial agent is prepared by the following method: (1) culture and enrichment of microbial bodies: Klebsiella sp. HNYJ-1 monoclone preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC No. 24327 is inoculated into LB liquid culture medium by using an inoculating loop, and is cultured to obtain original seeds; and (2) the flavoring and viscosity-reducing microbial agent is obtained by gradient induction of the microbial bodies. The flavoring and viscosity-reducing microbial agent is used for preparing reconstituted tobacco concentrate with flavoring and viscosity reduction. After treatment of the reconstituted tobacco concentrate with the flavoring and viscosity-reducing microbial agent, the contents of protein, pectin and starch in the reconstituted tobacco concentrate are reduced, the fiber is basically unchanged, and the viscosity is reduced; meanwhile, the contents of alcohol, aldehyde ketone and ester flavoring substances in the reconstituted tobacco concentrate are obviously increased, the content of heterocyclic substances is increased, and the total amount of flavoring substances is increased.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a flavor-enhancing and viscosity-reducing bacterial agent and its application in reconstituted tobacco concentrate. Background Technology

[0002] Reconstituted tobacco using the papermaking method is a tobacco product made from tobacco materials (stems, dust, and broken tobacco leaves) as the main raw material. Through processes such as extraction, pulping, concentration, papermaking, coating, and drying, it plays a crucial role in the development of cigarette brands and the stable improvement of cigarette quality. After water extraction, the extract is concentrated to form a concentrate. The solids are then compressed into sheets, and the concentrated tobacco solution is uniformly coated onto the sheets to produce reconstituted tobacco. The sheets provide the skeletal structure of the reconstituted tobacco, while the concentrate provides the main aroma components. However, currently, most reconstituted tobacco processing concentrates suffer from two main problems: firstly, insufficient aroma components; and secondly, high viscosity, resulting in low sheet coating rates and potential equipment blockage, failing to meet the higher processing requirements and product quality demands of reconstituted tobacco manufacturers.

[0003] The aroma compounds in the concentrate originate from tobacco substances. During the preparation process, low extraction rates and losses due to high-temperature concentration result in insufficient aroma compound content. The high viscosity of the concentrate is mainly due to the high content of macromolecules such as polysaccharides, pectin, starch, and proteins. Reducing the content of macromolecules can effectively reduce the viscosity of the concentrate. In actual production processes, numerous experiments have been conducted to address this issue: adding external flavorings to improve the aroma is costly and generally not well-coordinated; improving material conveying equipment and using silicon carbide ultrafiltration membranes to purify and reconstitute the tobacco concentrate can solve the viscosity problem, but the actual results are not ideal, and the root cause of the problem has not been addressed.

[0004] Microbial degradation of macromolecules is characterized by its green and efficient nature. Selecting suitable microorganisms to treat concentrates can, on the one hand, degrade macromolecules to reduce viscosity, and on the other hand, transform macromolecules into aroma components. Currently, there are few reports on using microorganisms to enhance the aroma and reduce the viscosity of concentrates in the papermaking reconstituted tobacco process. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an aroma-enhancing and viscosity-reducing bacterial agent and its application in reconstituted tobacco concentrate, which overcomes the above-mentioned defects of the prior art.

[0006] The technical problem to be solved by the present invention is achieved through the following technical solution:

[0007] A flavoring and viscosity-reducing bacterial agent, said flavoring and viscosity-reducing bacterial agent is prepared by the following method:

[0008] (1) Culture and enrichment of bacterial cells:

[0009] A single colony of Klebsiella sp. HNYJ-1, deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 24327, was inoculated into LB liquid medium using an inoculation loop and cultured in a shaker at 25-40℃ and 100-200 r / min for 6-36 h to obtain the original seed.

[0010] (2) Obtaining aroma-enhancing and viscosity-reducing bacterial agents by bacterial gradient induction:

[0011] (21) Select a concentrate with a viscosity of 82cp±0.5cP, dilute it with pure water to obtain a concentrate with a volume ratio of 30% and 70%;

[0012] (22) Centrifuge the original seed with a volume of V1 to remove the supernatant. After the bacterial cells precipitate, place them all in a 30% concentrated solution with a volume of V1. Incubate in a shaker at 25-40℃ and 100-200r / min for 6-36h. After centrifugation, remove the supernatant and dissolve it in deionized water with a volume of 80% V1 to obtain the primary induction seed. Divide it into two equal parts, one part for later use and the other part for further induction.

[0013] (23) One portion of the primary induction seeds was placed in a 70% concentrate of volume V2 and cultured in a shaker at 25-40℃ and 100-200r / min for 6-36h. After centrifugation, the supernatant was removed and dissolved in deionized water of volume 80% V2 to obtain secondary induction seeds. The seeds were then divided into two equal portions, one portion was kept for later use and the other portion was used for further induction.

[0014] (24) One portion of the secondary induction seeds was placed at 4℃ and allowed to stand for 6-48 hours. Then it was mixed with 100% concentrated solution (V3 volume) and cultured in a shaker at 25-40℃ and 100-200 r / min for 6-36 hours. After centrifugation to remove the supernatant, the seeds were dissolved in 80% deionized water (V3 volume) to obtain tertiary induction seeds.

[0015] The primary induced seeds, the secondary induced seeds, and the tertiary induced seeds are aroma-enhancing and viscosity-reducing bacteria.

[0016] Preferably, in step (1), the culture conditions for the original seeds are: 30°C and 150 r / min in a shaker for 12 h.

[0017] Preferably, in step (2), the culture conditions for the primary induction seeds are: 30℃ temperature and 150r / min shaking culture for 24h, the culture conditions for the secondary induction seeds are: 30℃ temperature and 150r / min shaking culture for 18h, and the culture conditions for the tertiary induction seeds are: 30℃ temperature and 150r / min shaking culture for 12h.

[0018] Preferably, in step (2), V2 / V1 = V3 / V2 = 40%.

[0019] A method for preparing aroma-enhancing and viscosity-reducing reconstituted tobacco concentrate using aroma-enhancing and viscosity-reducing bacteria includes the following steps:

[0020] Add 2% (v / v) of third-stage induction seeds to the reconstituted tobacco concentrate, set the temperature to 25-40℃, and stir at 50-200 r / min for 6-36 h. After fermentation, an aroma-enhanced and viscosity-reducing reconstituted tobacco concentrate is obtained; or

[0021] Add 0.5-10% (v / v) of primary induction seeds and 0.1-5% (v / v) of secondary induction seeds to the reconstituted tobacco concentrate. After mixing evenly, set the temperature to 25-40℃ and the stirring speed to 50-150 r / min for 6-36 h. Then add 0.1-5% (v / v) of tertiary induction seeds, set the temperature to 25-40℃ and the stirring speed to 50-200 r / min and continue to ferment for 6-36 h. After fermentation, the reconstituted tobacco concentrate with enhanced aroma and reduced viscosity is obtained.

[0022] Application of an aroma-enhancing and viscosity-reducing agent in reconstituted tobacco concentrate. After treatment with the aroma-enhancing and viscosity-reducing agent, the protein and starch content in the prepared concentrate decreased, while pectin and fiber remained essentially unchanged, and viscosity decreased. After treatment with the aroma-enhancing and viscosity-reducing agent, the content of alcohols, aldehydes, ketones, and esters in the reconstituted tobacco concentrate increased significantly, heterocyclic compounds increased, and the total amount of aroma substances improved.

[0023] Application of a reconstituted tobacco concentrate with enhanced aroma and reduced viscosity in tobacco leaves.

[0024] The above-described technical solution of the present invention has the following beneficial effects:

[0025] (1) Klebsiella pneumoniae HNYJ-1 can specifically degrade proteins and pectins, thereby reducing the viscosity of reconstituted tobacco concentrate. After treatment with aroma-enhancing and viscosity-reducing bacteria, the starch content in the reconstituted tobacco concentrate decreased, while the fiber content remained essentially unchanged, and the viscosity decreased.

[0026] (2) The overall quality of reconstituted tobacco concentrate was significantly improved after treatment with aroma-enhancing and viscosity-reducing bacteria. After treatment with aroma-enhancing and viscosity-reducing bacteria, the content of alcohols, aldehydes, ketones and esters in the reconstituted tobacco concentrate increased significantly, heterocyclic substances increased, and the total amount of aroma substances increased. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. It should be noted that, unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the invention.

[0028] This application utilizes Klebsiella sp. HNYJ-1 to prepare a flavoring and viscosity-reducing bacterial preparation. The flavoring and viscosity-reducing bacterial agent used is Klebsiella sp. HNYJ-1, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24327 and deposit date of January 17, 2022.

[0029] Example 1: Culture and enrichment of bacterial cells

[0030] Single colonies of Klebsiella pneumoniae (HNYJ-1) were inoculated into LB liquid medium using an inoculation loop and cultured in a shaker at 25-40℃ (optimal 30℃) and 100-200 r / min (optimal 150 r / min) for 6-36 h (optimal 12 h) to obtain the original seed.

[0031] Example 2 Gradient Induction of Bacterial Cells

[0032] (1) Select a concentrate with a viscosity of 82cp±0.5cp (poise) and dilute it with pure water to obtain a concentrate with a volume ratio of 30% and 70%.

[0033] (2) Centrifuge the original seed with a volume of V1 to remove the supernatant. After the bacterial cells precipitate, place them all in a 30% concentrated solution with a volume of V1. Incubate in a shaker at 25-40℃ (optimal 30℃) and 100-200r / min (optimal 150r / min) for 6-36h (optimal 24h). After centrifugation, remove the supernatant and dissolve it in deionized water with a volume of 80% V1 to obtain the primary induction seed. Divide it into two equal parts, one part for later use and the other part for further induction.

[0034] (3) Take one portion of the primary induction seeds and place it in a 70% concentrate of volume V2. Incubate it in a shaker at 25-40℃ (optimal 30℃) and 100-200r / min (optimal 150r / min) for 6-36h (optimal 18h). After centrifugation, remove the supernatant and dissolve it in deionized water of volume 80% V2 to obtain secondary induction seeds. Divide the seeds into two equal portions, keep one portion for later use, and continue induction with the other portion.

[0035] (4) One portion of the secondary induction seeds was placed statically at 4℃ for 6-48 hours (optimal 18 hours), then mixed with 100% concentrated solution (undiluted concentrate) at volume V3, and cultured in a shaker at 25-40℃ (optimal 30℃) and 100-200 r / min (optimal 150 r / min) for 6-36 hours (optimal 12 hours). After centrifugation to remove the supernatant, the seeds were dissolved in 80% V3 of deionized water to obtain tertiary induction seeds. (Where V2 / V1 = V3 / V2 = 40%)

[0036] Example 3: Fermentation treatment of reconstituted tobacco concentrate

[0037] Option 1 (adding only a level 3 induction seed):

[0038] Add 2% by volume of third-stage induction seeds to the reconstituted tobacco concentrate, and set the temperature to 25-40℃ (optimal 30℃) and the speed to 50-200r / min (optimal 150r / min) for stirring and fermentation for 6-36h (optimal 12h).

[0039] After fermentation, a concentrated reconstituted tobacco leaf extract with enhanced aroma and reduced viscosity is obtained.

[0040] Option 2 (selecting to add three levels of induction seeds step by step):

[0041] (1) Add 0.5-10% (optimal 1%) of primary induction seeds and 0.1-5% (optimal 0.5%) of secondary induction seeds to the reconstituted tobacco concentrate. After mixing evenly, set the temperature to 25-40℃ (optimal 30℃) and the speed to 50-150r / min (optimal 100r / min) and stir for fermentation for 6-36h (optimal 12h).

[0042] (2) Add 0.1-5% (optimal 0.5%) of third-stage induction seeds, set the temperature to 25-40℃ (optimal 30℃) and 50-200r / min (optimal 150r / min) and continue stirring and fermenting for 6-36h (optimal 12h).

[0043] After fermentation, a concentrated reconstituted tobacco extract with enhanced aroma and reduced viscosity is obtained. No sterilization is required after fermentation; it can be directly used for coating. The high-temperature process in the reconstituted tobacco forming process achieves sterilization.

[0044] Application Example 1

[0045] The TS-006 (intermediate flavor) reconstituted tobacco concentrate from Henan Tobacco Industry Tobacco Sheet Co., Ltd. was selected as the test sample.

[0046] (1) Set the unfermented reconstituted tobacco concentrate as CK, and add 2% deionized water by volume to the reconstituted tobacco.

[0047] (2) Add 2% of the volume fraction of the third-level induction seeds to the reconstituted tobacco concentrate, set the temperature to 30℃, stir and ferment at 150r / min for 12h, and the reconstituted tobacco concentrate after fermentation is T1.

[0048] (3) Add 1% by volume of primary induction seeds and 0.5% by volume of secondary induction seeds to the reconstituted tobacco concentrate, mix well, and ferment at 30℃ and 100r / min for 12h. Then add 0.5% by volume of tertiary induction seeds, and continue fermenting at 30℃ and 150r / min for 12h. The treated reconstituted tobacco concentrate is recorded as T2.

[0049] Results analysis:

[0050] The protein, pectin, starch, and cellulose content and viscosity of the reconstituted tobacco concentrate were measured, and the results are shown in Table 1. The content of neutral aroma components in the reconstituted tobacco concentrate was also measured, and the results are shown in Table 2.

[0051] After treatment with the aroma-enhancing and viscosity-reducing bacteria, the content of protein, pectin, and starch in the reconstituted tobacco concentrate was significantly reduced, while the fiber content remained essentially unchanged, and the viscosity decreased markedly. This demonstrates that Klebsiella pneumoniae HNYJ-1 can specifically degrade protein, pectin, and starch, thereby reducing the viscosity of the reconstituted tobacco concentrate.

[0052] After treatment with aroma-enhancing and viscosity-reducing bacteria, the content of alcohols, aldehydes, ketones, and esters in the reconstituted tobacco concentrate increased significantly, while heterocyclic compounds slightly increased, resulting in an overall improvement in the total amount of aroma compounds. This demonstrates that treatment with aroma-enhancing and viscosity-reducing bacteria significantly improves the overall quality of the reconstituted tobacco concentrate.

[0053] The improvement effect of T2 tobacco leaves treated with three levels of inducing seeds is better than that of T1 tobacco leaves treated with only three levels of inducing seeds. However, the improvement effect of T1 tobacco leaves is not bad (only slightly inferior to that of T2 tobacco leaves). Therefore, the inducing seed addition scheme can be selected according to specific needs: adding only three levels of seeds is simple and cost-effective, while adding one, two, and three levels of inducing seeds at the same time is more complex, but the treatment effect is better.

[0054] Table 1. Changes in macromolecular substances and viscosity of reconstituted tobacco concentrate before and after fermentation.

[0055]

[0056] Note: Viscosity is expressed in poise (cP) (1 cP = 1 mPa·s).

[0057] Table 2. Analysis of major aroma components in reconstituted tobacco concentrate (content μg / mL)

[0058] sample alcohols Acids Aldehydes and ketones esters Phenols Heterocyclic total CK 468.27 108.65 276.78 79.68 121.18 53.38 1107.94 T1 512.45 113.86 343.58 182.63 107.82 56.02 1316.36 T2 539.83 125.22 353.89 207.38 105.37 71.08 1402.77

[0059] Although the present invention has been disclosed above with reference to embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various different choices and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention is defined by the claims and their equivalents.

Claims

1. A fungicidal bacteriostatic agent, characterized by comprising a mixture of a volatile organic compound and a volatile inorganic compound. The flavoring viscosity-reducing microbial agent is prepared by the following method: (1) Cultivation and enrichment of the microbial bodies: Klebsiella (Klebsiella sp.) HNYJ-1 preserved in China General Microbiological Culture Collection Center with the preservation number of CGMCC No. 24327 Klebsiella sp. The single colony of Klebsiella (Klebsiella sp.) HNYJ-1 is inoculated into LB liquid medium by an inoculating ring, and cultured at 25-40 °C in a shaker at 100-200 r / min for 6-36 h to obtain the original seed. (2) Gradient induction of the microbial bodies to obtain the flavoring viscosity-reducing microbial agent: (21) The concentrated solution with a viscosity of 82 cp±0.5 cP is diluted with pure water to obtain concentrated solutions with a volume ratio of 30% and 70%; (22) The original seed with a volume of V1 is centrifuged to remove the supernatant, and the microbial body precipitate is placed in the 30% concentrated solution with a volume of V1. The mixture is cultured at 25-40℃ and 100-200 r / min for 6-36 h in a shaker. After centrifugation, the supernatant is removed, and the microbial body is dissolved in deionized water with a volume of 80% V1 to obtain the first induced seed. The first induced seed is equally divided into two parts, one of which is reserved for standby, and the other is continuously induced; (23) One of the first induced seeds is mixed with the 70% concentrated solution with a volume of V2, and the mixture is cultured at 25-40℃ and 100-200 r / min for 6-36 h in a shaker. After centrifugation, the supernatant is removed, and the microbial body is dissolved in deionized water with a volume of 80% V2 to obtain the second induced seed. The second induced seed is equally divided into two parts, one of which is reserved for standby, and the other is continuously induced; (24) One of the second induced seeds is placed at 4℃ for 6-48 h, and then mixed with the 100% concentrated solution with a volume of V3. The mixture is cultured at 25-40℃ and 100-200 r / min for 6-36 h in a shaker. After centrifugation, the supernatant is removed, and the microbial body is dissolved in deionized water with a volume of 80% V3 to obtain the third induced seed. The first induced seed, the second induced seed, and the third induced seed are the flavoring viscosity-reducing microbial agent.

2. The viscosity-reducing and odor-improving bacterial agent according to claim 1, characterized by, In step (1), the original seed is cultured at a temperature of 30℃ and a rotation speed of 150 r / min for 12 h in a shaker.

3. The viscosity-reducing and odor-improving bacterial agent according to claim 1, characterized by, In step (2), the first induced seed is cultured at a temperature of 30℃ and a rotation speed of 150 r / min for 24 h in a shaker, the second induced seed is cultured at a temperature of 30℃ and a rotation speed of 150 r / min for 18 h in a shaker, and the third induced seed is cultured at a temperature of 30℃ and a rotation speed of 150 r / min for 12 h in a shaker.

4. The viscosity-reducing and odor-improving bacterial agent according to claim 1, characterized by, In step (2), V2 / V1=V3 / V2=40%.

5. A method for preparing flavoring and viscosity reducing reconstituted tobacco concentrate using the flavoring and viscosity reducing bacterial agent according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: The first induced seed with a volume fraction of 0.5-10%, the second induced seed with a volume fraction of 0.1-5%, and the third induced seed with a volume fraction of 0.1-5% are added to the reconstituted tobacco concentrate, and the mixture is uniformly mixed. Then, the temperature is set to 25-40℃, the stirring speed is set to 50-150 r / min, and the mixture is fermented for 6-36 h. Then, the third induced seed with a volume fraction of 0.1-5% is added, the temperature is set to 25-40℃, and the stirring speed is set to 50-200 r / min for continuous fermentation for 6-36 h. After the fermentation is completed, the reconstituted tobacco concentrate with reduced viscosity and flavor is obtained.

6. The method of claim 5, wherein, After the reconstituted tobacco concentrate is treated by the flavoring viscosity-reducing microbial agent, the contents of protein, pectin, and starch in the reconstituted tobacco concentrate are reduced, the fiber content is basically unchanged, and the viscosity is significantly reduced.

7. The method of claim 5, wherein, After the reconstituted tobacco concentrate is treated by the flavoring viscosity-reducing microbial agent, the contents of alcohol, aldehyde ketone, and ester flavoring substances in the reconstituted tobacco concentrate are significantly increased, the content of heterocyclic substances is slightly increased, and the total amount of flavoring substances is increased.

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